Glycosyl pantothenic acids

ABSTRACT

A GLYCOSYL PANTOTHENIC ACID HAVING A FORMULA OF   R1-O-CH2-C(-CH3)2-CH(-O-R2)-CO-NH-CH2-CH2-COOH   WHEREIN ONE OF R1 AND R2 IS HYDROGEN AND THE OTHER IS B-RIBOFURANOXYL, B-D-GLUCOPYRANOSYL, B-D-GALACTOPYRANOSYL, B-CELLOBIOSYL OR B-MALTOSYL: THIS COMPOUND HAS HIGHER PHYSIOLOGICAL ACTIVITY THAN THAT OF CALCIUM DPANTOTHENIC ACID.

United States Patent Oflice 3,808,196 Patented Apr. 30, 1974 3,808,196GLYCOSYL PANTOTHENIC ACIDS Hajime Yoshizumi, Tondabayashi, Teruo Amachi,Takarazuka, Shoji Imamoto, Kishiwada, and Siro Senoh, Nishinomiya,Japan, assignors to Suntory Limited, Osaka-shi, Japan No Drawing. FiledOct. 22, 1971, Ser. No. 191,851 Claims priority, application Japan, Oct.22, 1970, 45/913,332, 45/915,333 Int. Cl. C07c 47/18 US. Cl. 260-210 R 4Claims higher physiological activity than that of calcium D- pantothenicacid.

This invention relates to novel glycosyl pantothenic acids and to aprocess for manufacturing the same.

The glycosyl pantothenic acids of the present invention are representedby the formula of wherein one of R and R is hydrogen and the other is oD ribofuranosyl, ,B-D-glucopyranosyl, ,8-Dgalactopyranosyl,p-cellobiosyl or B-maltosyl. The present compounds are novel and includetwo types of compounds, i.e., 2'-O-(fi-glycosyD-D-pantothenic acidshaving the following Formula LA and 4-O-(fi-glycosyl)-O-panto-.

thenic acid having the Formula I-B.

wherein R and R are B-D-ribofuranosyl, fl-D-glucopyranosyl,fl-D-galactopyranosyl, [i-cellobiosyl or fi-maltosyl.

Both types of compounds are hygroscopic powder and soluble in water,methanol, ethanol, pyridine, etc., but insoluble in benzene, ethylether, chloroform, etc.

The present compounds are derivatives of D-pantothenic acid and havephysiological activity like D-pantothenic acid. For example, theydisplay adjusting effect on metabolism in microorganisms. Thephysiological activity of the present compounds is superior to that ofcalcium D- pantothenate. For instance, they display several times to onehundred times the activity of calcium D-pantothenate on strainsbelonging to the genus lactobacillaceae. Furthermore, the presentcompounds are more excellent in alkali-resistant, acid-resistant andheat-resistant properties as compared with D-pantothenic acid.Accordingly the present compounds are useful for various applications asmedicines, cosmetic, food additives, additives for feeds, as medicines,cosmetics, food additives, additives for feeds, additives for culturemedium for microorganisms, etc.

2-O-(fi-D-glucopyranosyl)-D-pantothenic acid,2-O-(B-cellobiosyl)-D-pantothenic acid,4'-O-(fl-D-glucopyranosyl)D-pantothenic acid, 4-O-(fl-D-ribofuranosyl)-D-pantothenic acid,4'-O-(B-D-galactopyranosyl)-D-pantothenic acid,4'-O-(/S-cellobiosyl)-D-pantothenic acid and4'-O-(fi-maltosyD-D-pantothenic acid.

Of these compounds, 4-O-( fl-D-glucopyranosyl)-D- pantothenic acid isthe most preferable, because it displays higher order of physiologicalactivities for some microorganisms.

To clarify the physiological activities of the present compounds thefollowing tests were per-formed.

(I) PHYSIOLOGICAL ACTIVITY ON LACTOBACILLACEAE Test A.--Activity of 4' O(B D-glucopyranosyl)-D- pantothenic acid on WNB- strain A-1. Basalmedium (double strength) Wt./v. percent Dglucose 1.0 D-fructose 1.0Yeast extract 1.0 Polypeptone 1.0 KH PO, 0.1 NaCl 0.025 CaCl -2H O 0.025MgSO -7H O 0.025 KCl 0.025 MnSO, 0.006

These components were dissolved in deionized water and the pH of theresulting solution was adjusted to 5.0. The solution thus prepared wasused as a basal medium.

A-2. Test method-To the basal medium was added4-O-(fi-D-glucopyranosyl)-D-pantothenic acid in the amount given inTable 1, and then the medium was sterilized at C. for 5 minutes. Thesurface 'of the medium was then covered with liquidv parafiine. WNB- 75strain was inoculated on the medium and incubated at 30 C. for 4 days.The number of the cells grown was measured by a colony count on agarplate with the lapse of time. The WNB-75 strain is a known strainbelonging to lactobacillaceae and described in Agr. Biol. Chem, Vol.27,No. 8, pages 590-595 (1963). The results are given in Table 1. Forcomparison, calcium D-pantothenate was addedto the same basal medium asabove and the strain was cultured in the same manner as above. Theresults are also set forth in Table 1.

A-3. lResults.- J. Enol. Viticult., vol. 11, 1-4 (1960) by I. L.Ingraham,

TABLE 1 Incubation time (hr.) at 30 0.

Amount 24 48 72 96 added Compound tested (pg/ml.) Number of cells(cells/ml.)

Control 0 10 7 8 10 3 Present compound 0. 0 10 X10 3x10 1. 4x10 4x10 0.1 7X10 4X10 6X10 5. 6X10 Calcium D-pantothenate 0. 5 10 2x10 8x10 1.5X10 6X10 5. 0 10 1. 3X10 2X10 9X10 3X10" The test results show that4'-O-(p-D-glucopyranosyD- R. H. Vaughn and G. M. Cooke and vol. 11,59-63 D-pantothenic acid is capable of effecting growth of (1960), by R.B. Webb and]. L. Ingraham.

WN'B75 strain in a concentration of 1/100 that of cal- C-2. Results.

TABLE 3 Incubation time (hr.) at C.

Amount 0 24 4s 72 96 added Compound tested g/ml.) Number of cells(cells/ml.)

Control 0 10 15 7 11 6 Present compound.-. 0.05 10 9x10 5x10 1.8)(10 710 0.1 10 4x10 1x10 5x10 0x10 Calcium D-pantothenate 0.5 10 3X10 8X101.1X10 2X10 5.0 10 5 10 1.7 10 2x10 4x10 cium -D-pantothenate. In otherwords, the former has an (II) PHYSIOLOGICAL ACTIVITIES ON RAT act1v1ty100 t1mes that of the latter. 30 Test method Test B'A.ctiviti?s of g gfof thls A powdery assorted feed CA-1 (trademark, product mventlon on n 5mm of Japan Clea Co.) containing 80 mg./ kg. of calcium D- B-l. Testmethod.-Each of several compounds of pantothenate was fortified with 1mg. and 5 mg. of 4'-O- this invention listed below was added to the samebasal (fl-D-glucopyranosyl)-D-pantothenic acid per kilogram of medium asused for Test A and WNB-75 strain was culthe feed. Another two kinds offeed were prepared for tured in the same manner as in Test A todetermine the comparison by further fortifying the same feed (CA-1)physiological activities of the present compounds relative with 50 mg.and 100 mg. of calcium D-pantothenate per to those of calciumD-pantothenate. The physiological kilogram of the same. Each of thefeeds thus prepared was activities of the test compounds were expressedin terms fed to 10 male, three-Week old SD-JCL rats. The rats ofrelative activities comparing with the activity of cal- 40 were free totake the feed and water, and the weight of the cium D-pantothenate whichis defined as one unit. rats was measured every day.

The results are given in Table 2. The average weight of the 10 rats ineach group is given B-2. Results.- I in Table 4.

TABLE 4 Amount Days elapsed after initiation of added experimentPercent;

(mg-[kgrelative Compound tested d1et 0 10 20 30 to control Control 48100 194 292 100 Present compound 1 48.5 126.5 221.5 328 112. 33

Calcium D-pantothenate-.- 50 47.5 111.5 199.5 292.5 100.17

TABLE 2 The results of the experiment show that the weight in- Compoundtested: Activity (unit/D-PaA) crease, for instance, in the case of 1mg./kg. diet of the Calcium D-pantotheuate 1 present compound was 36 g.greater (i.e. increase of 2'-O-(fi-D-glucopyranosyl)-D-pantothenic acid5 12.3%) on average than the control. This exceeds the2'-'O-(B-cellobiosyl)-D-pantothenic acid 20 result achieved with a lotfortified by calcium D-panto- 4'-0-(fl-D-galactopyranosyl)-D-pantothenicacid 5 thenate, and shows that the present compound can be sub-4'-O-(p-D-cellobiosyl)-D-pantothenic acid 20 stituted for calciumD-pantothenate in a smaller amount Tast C PhySio1ogica1 activities onLeuconosmc than is the case with calcium .D-pantothenate. Thecondicl-tmvomm ML 34 strain tion of the whole lot was normal andhealthy.

The present compounds can be prepared by various Test method--30 theSame basal medium as used methods. \According to one of the preferredmethods for in Test A was adde -(l D j fpy y preparing the compoundrepresented by the Formula I-1A pantothemc q m the amount llsfed m Table3, and before, alkyl 4'-O-acyl-Dpantothenate represented by theLeuconostoc cltrovorum ML-34 stram was cultured in the F l 11 below, forxample, is reacted with a halide same manner as in Test A to determinethe number of of acyl sugar to produce an intermediate, alkyl 4'-O- 1-grown cells. The results are given in Table 3, which for2'-O-(O-acyl-fi-glycosyl)-D pantothenate represented by comparison,further includesfest results obtained in the the Formula III below(hereinafter referred to as intersame manner as above by usmg calciumD-pantothenate. mediate A and the intermediate A thus obtained is Theabove-mentioned Leuconostoc citrovorum 1s a known then subjected todeacylation and dealkylation reaction to strain belonging to lactobacilaceee an disclosed n Am p duce the desired Z-O-(p-gIycOsyI) Danthothenic acid (I-A). This method is hereinafter referred to as MethodA and the reactions involved therein are shown by the following reactionequations:

CH: .AcOCHa-(JCHC ONHCHaCHrCOOR OH: OH

CHa +AcR .X A) AGOCHn- CHC ONHCH CH COOR CH: R-Ac Deaeylatlon and CH:dealkylation (E HOCHg- CHC ONHCHzCHaC O OH CH3 R wherein R is the sameas defined before, Ac is acyl group such as acetyl or benzoyl, X is ahalogen atom and R is an alkyl group of l to 4 carbon atoms.

In the above Method A the alkyl 4'-O-acyl-D-pantothenate (II) used as astarting material is known in the art and easily prepared by thermalcondensation of 2-0- benzyl-D-pantolactone and ,B-alanine alkyl ester,acylation of hydroxyl group at C-4 of the resultant condensation productand then debenzylation with catalytic hydrogenation. The above alkylesters of acyl pantothenic acid (II) include methyl, ethyl, propyl,isopropyl and butyl esters, the preferable being ethyl ester. Thehalides of acyl sugars used include chlorides or bromides of acylderivatives of D-ribofuranose, D-glucopyranose, D-galactopyranose,D-cellobiose or D-maltose.

The reaction of the alkyl esters of acyl pantothenic acid (II) withhalide of acyl sugar in the first step can be conducted in the solventsuch as nitromethane, benzene, toluene, chloroform, etc. The halide ofacyl sugar is used in a stoichiometric amount or in an excess amount,preferably in an amount to 50% in excess. The solvent may be used singlyor in admixture with one another. The amount of the solvent used is inthe range of 10 to 50 times the weight of the starting esters (II). Toneutralize the hydrogen halide formed, an acid acceptor may be added tothe reaction system, if desired. Examples of the acid acceptors areHg(CN) Ag CO AgO', AgClO AgBF etc., the preferable being Hg(CN) Theamount of the acid acceptor used may vary over a wide range but usuallyit is employed in an amount ranging from an equimolar amount to agreatly excessive amount based on the mole of the halide of acyl sugar.Preferable reaction temperature is in the range of 80 to 110 C., thoughthe reaction proceeds at a lower temperature with a retarded rate and ata higher temperature with undesired side reaction. To the reactionsystem there may be added as a reaction promotor dehydrating agents suchas CaSO etc., if desired. The promotor is used in an amount of l to 10times the weight of the starting ester (H). It is preferable to carryout the reaction in an atmosphere free of water vapor, for example, indried air or inert gas atmosphere. In general the reaction completeswithin about 5 to 20 hours, whereby the intermediate A alkyl4-Oacyl-2'-O'-(O-acyl-flglycosyl)-D-pantothenate (III) can be obtained.The intermediate A can be isolated, as required, from the resultantreaction mixture, for example, by filtrating the reaction mixture,concentrating the filtrate under reduced pressure, dissolving theconcentrated mixture in benzene or other nonpolar solvents, washing thebenzene solution with aqueous alkali metal halide solution and then withwater, concentrating the washed mixture and subjecting the concentratedmixture to silica gel column chromatography using a 1:1 volume ratiomixture of benzene and ethyl acetate.

The intermediate A can be subjected to subsequent deacylation anddealkylation reaction as it is in the reaction mixture resulting fromthe first-step reaction or after it is isolated. The second-stepreaction, i.e., deacylation and dealkylation reaction of theintermediate A (III) thus obtained is conducted in an organic solventsuch as methanol, ethanol or like lower aliphatic alcohol in thepresence of a catalyst. The solvent may be employed in an amount of 5 to10 times the weight of the intermediate A Examples of the catalysts areBa(OCH NaOCH NH Ba(OH) etc. and they can be used in an amount of alittle molar excess, preferably one tenth excess of the intermediate AIt is preferable to carry out the reaction at a temperature of 0 to 30C., as undesired side reaction is promoted at a higher temperature. Thereaction can be conducted in an atmosphere free of water vapor such asin dried air or an inert gas atmosphere. The reaction usually completeswithin 1 to 24 hours in accordance with the reaction conductionsapplied, whereby the desired 2'-O-(B-glycosyl)-D-pantothenic acid (IA)is obtained. The desired product (I-A) thus obtained can be isolated,for example, by adding water to the resultant reaction mixture, passingthe mixture through a column of OH-form anion exchange resin, such asDowex l x 8" (trademark, the Dow Chemical Co., U.S.A.), washing theion-exchanged substance with water, eluting the substance with aneluent, such as acetic acid, and freeze-drying the solution to producethe desired product (I-A) in the form of a white hygroscopic powder.

Another type of the product, 4'-O-( 3-glycosyl)-D- pantothenic acid ofthe invention represented by the formula of LB can also be prepared byvarious methods. According to one of the preferred methods benzyl 2-O-benzyl-D-pantothenate having the Formula IV below is first reacted withhalide of acyl sugar to produce benzyl 2'-O-benzyl 4O-(O-acyl-B-glycosyl)-D-pantothenate having the Formula V below(hereinafter referred to as intermediate B The intermediate B (V) isthen hydrogenated in the presence of catalyst to produce 4'-O-(Oacyl-B-glycosyl)-D-pantothenic acid having the formula of (VI)(hereinafter referred to as intermediate B and the intermediate B (VI)thus obtained is finally subjected to deacylation reaction, whereby thedesired 4'-O-(B-glycosyl)-Dpantothenic acid having the Formula I-B canbe obtained. The above method is hereinafter referred to as Method 1Band reactions involved therein are shown by the following equations:

C Ha noon, CCHC oNflomomo 00B z on, DB2

CH3 AcR X BAAcOCH CHCONHCHCH;COOBZ CH3 +H2 (IJ Rue-00H.-CHCONHCH2CH2COOH OH: OH

CH3 Deacylatlon (1J R 003. CHCONHCH2CH:COOH

Hz OH wherein Bz shows benzyl group and R Ac and X are the same asdefined before.

In the above Method 1B the benzyl. 2'-O-benzyl-D- pantothenate (IV) usedas a starting material is known in the art and can be prepared, forexample, by the thermal condensation of Z-O-benzyl-D-pantolactone andfl-alanine benzyl ester. The halides of acyl sugars used are the same asin the Method A before.

In the Method 1B the reaction between benzyl 2'-O- benzyl-D-pantothenate(IV) and halide of acyl sugar can be conducted under the same conditionsas those disclosed in the first step of the Method A. The intermediate B(V) Obtained in this step can be isolated from the resultant reactionmixture, for example, by filtrating the reaction mixture, concentratingthe filtrate, dissolving the concentrated liquid in benzene, washing thesolution with aqueous potassium halide solution and then with saturatedsodium hydrogen carbonate solution and further with water, concentratingthe washed liquid under reduced pressure and finally subjecting theconcentrated liquid to silica gel column chromatography using chloroformcontaining 2.5 vol. percent of ethanol.

In the second step the intermediate B (V) is dissolved in an organicsolvent and the solution is stirred in the presence of a catalyst in ahydrogen gas atmosphere for reduction reaction. The solvents usedinclude acetic acid, methanol, ethanol, etc. These solvents can beemployed alone or in admixture with one another in an amount of 2 to 10times the weight of the intermediate B In the above reactionconventional reduction catalysts such as palladium black, palladiumcharcoal, platinium oxide etc. can be used in an amount of l to 100weight percent, based on the weight of the intermediate B Preferably thereaction be carried out at room temperature in a hydrogen atmosphere,though elevated temperatures and/ or increased pressures may also beapplicable. The reaction usually completes within 5 to 10 hours, wherebythe intermediate B having the Formula VI can be obtained. Theintermediate B (VI) thus obtained can be isolated from the resultantreaction mixture, for example, by subjecting the reaction mixture tosilica gel column chromatography using a 95:4:1 volume ratio mixture ofCHCl CH OH and CH COOH or a 95:5 volume ratio mixture of CHCl and C H OHand then recrystallizing the resultant substance from ether.

In the third step the intermediate B is deacylated to produce thedesired Compound I-B. The deacylated conditions are the same as thosedisclosed in deacylation and dealkylation conditions in the second stepof the Method A before.

The 4-O-(,6-glycosyl)-D-pantothenic acid (I-B) thus obtained can beisolated from the reaction mixture in the same manner as in the case ofthe Compound I-A obtained by the Method A before.

According to another preferred method for preparing4-O-(B-glycosyl)-D-pantothenic acid (I-B) of the invention alkylD-pantothenate having the -Formula VII below is first reacted withhalide of acyl sugar to produce alkyl4'-O-(O-acyl-fi-glycosyl)-D-pantothenate having the Formula VIII belowand the resultant alkyl 4'-O-(O-acyl-[3- glycosyl)-D-pantothenate (VIII)is then subjected to deacylation and dealkylation reaction, whereb thedesired 4'-O-(p-glycosyl)-D-pantothenic acid (I-B) is obtained. Thismethod is hereinafter referred to as Method 28" and reactions involvedtherein are shown by the following equations:

a HOCH: CCHC ONHCHzCI-Ia C R CH3 H (VII) CH3 AcR -X R4ACOCHZ!CICHCONHCHQCI'IQCOORG CH3 OH (VIII) Deacylatlon CH3 and dealkylation (5 ROCI-I OHCONHCH1CH;COOH

OH; H

wherein R Ac and X are the same as defined before and R is an alkyl of 1to 4 carbon atoms.

In the above Method 2B the alkyl D-pantothenate includes methyl, ethyl,propyl, isopropyl or butyl ester of D-pantothenic acid, and the halidesof acyl sugars used include chlorides and bromides of acyl derivativesof D- ribofuranose, D-glucopyranose, D-galactopyranose, D- cellobioseand D-maltose.

The reaction conditions to be applied in the first step reaction ofbetween alkyl D-pantothenate (VII) and halide of acyl sugar are the sameas disclosed relating to the reaction of between alkyl4'-O-acyl-D-pantothenate (II) and halide of acyl sugar in the Method Abefore. The resultant alkyl 4'-O-(O acyl-B-glycosyl)-D-pantothenate(VIII) can be isolated in the same manner as in the isolation of theintermediate A resulting from the first step of the Method A.

The subsequent deacylation and dealkylation of the alkyl 4'-O-(O-acyl ,Bglycosyl)-D-pantothenate can be carried out in the same manner as in thedeacylation and dealkylation of the intermediate A in the Method Abefore, whereby the desired 4'-O-(B-glycosyD-D-pantothenic acid (I-B)can be obtained. 4' O (B glycosyl)- D-pantothenic acid can be isolatedin the same manner as disclosed before.

For better understanding of the invention examples are given below.

EXAMPLE 1 2.9 g. of ethyl 4'-O-aceyl-D-pantothenate was dissolved in 20ml. of 2:1 volume ratio dried mixture of nitromethane and benzene. Tothe resultant solution were added 3.8 g. of dried Hg(CN) and 7.0 g. ofactivated CaSO in the form of powder and the resultant mixture wasstirred at room temperature for 1 hour. To the mixture was further added7.0 g. of tetra-O-acetyl-a-D-glucopyranosyl bromide and the mixture washeated at to 100 C. for 20 hours.

After being cooled, the resultant reaction mixture was filtered and thefiltrate was concentrated under reduced pressure. The concentratedliquid was dissolved in benzene to produce 100 ml. of benzene solutionand the solution was Washed with aqueous potassium bromide solution andthen with water. The washed liquid was evaporated to dryness underreduced pressure and thereafter subjected twice to silica gel columnchromatography using a 1:1 volume ratio mixture of benzene and ethylacetate, whereby 2.6 g. of ethyl4'-O-acetyl-2'-O'-(O-acetyle-D-glucopyranosyl)-D-pantothenate. Yield was42%.

The N.M.R. (nuclear magnetic resonance) in CD013 on the resultantproduct gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.92 (3H, s), 1.02 (3H, s), 1.30 (3H, t, J=7 c.p.s.), 2.0-

2.1 (5 x Me), 2.53 (2H, t, J=7 c.p.s.), 3.5-5.3 (In), 6.93 (1H, t).

1.3 g. of ethyl4'-O-acetyl-2'-O-(O-acetyl-/3-D-glucopyranosyl)-D-pantothenate thusobtained was dissolved in 30 m1. of dried methanol. To the solution wasadded 10 ml. of 0.4 N Ba(OCII solution dissolved in methanol while beingcooled with ice water and the resultant mixture was left to stand at 0C. for 24 hours. To the resultant reaction mixture was added 20 ml. ofwater and the mixture was passed through a column of DoweX 1 x 8(trademark, ion exchange resin of The Dow Chemical Co., U.S.A., used as0H form). After being Washed with water the product was eluted with 0.5N acetic acid and freeze-dried to produce 0.77 g. of 2-O-(fi-D-glucopyranosyl)-D-pantothenic acid in the form of whitehygroscopic powder. Yield was The N.M.R. spectrum on the resultantproduct in D 0 and elementary analysis thereof gave the followingresults.

N.M.R. spectrum (6, p.p.m.):

0.88 (6H, s), 2.58 (2H, t, 1:7 c.p.s.), 3.1-3.8 (In), 3.87

(1H, s), 4.38 (1H, (1, 1:6 c.p.s.).

Elementary analysis (percent): Found: C, 47.29; H, 7.06; 3.61. Calcd.for C H O N: C, 47.24; H, 7.14; N, 3.67.

EXAMPLE 2 To 30 ml. of benzene were added 2.9 g. of ethyl 4-O- acetyl Dpantothenate and 3.8 g. of dried Hg. (CN);; and the mixture was heatedwith stirring to evaporate 01f 20 ml. of benzene. Thereafter, 20 ml. ofdried benzene was further added to the mixture and after the benzene wasevaporated oil? with water as azeotropic mixture, 20 ml. of driednitromethane was further added thereto. To the resultant mixture wasadded 7.0 g. of O-acetyl a D- glucopyranosyl bromide and the mixture wasrefluxed with stirring for 20 hours. The resultant reaction mixture wassubjected to the same purification steps as in Example 1, whereby 1.3 g.of ethyl 4 O-acetyl-2'-O(O- acetyl-,B-D-glucopyranosyl)-D-pantothenatewas obtained. Yield was 21%.

The N.M.R. on the resultant product gave the same results shown as inExample 1.

From the resultant product was hydrolyzed and isolated by the sameprocedure as in Example 1, and 2-O- D glucopyranosyl) D pantothenic acidwas obtained. The N.M.R. on the product gave the same results as inExample 1.

EXAMPLE 3 To 30 m1. of a 2:1 volume ratio dried mixture of nitromethaneand benzene were added with stirring 2.9 g. of ethyl4-O-acetyl-D-pantothenate, 3.8 g. of Hg. (CN) and 7.0 g. ofO-acetyl-a-D-glucopyranosyl bromide. The resultant mixture was refluxedwith stirring for 24 hours. The resultant reaction mixture was subjectedto the same purification steps as in Example 1, whereby 0.19 g. of ethyl4-O-acetyl 2' O-(O-acetyl-p-D-glucopyranosyl)- D-pantothenate wasobtained.

The N.M.R. on the resultant product gave the same results shown inExample 1.

The resultant product was hydrolyzed and isolated in the same manner asin Example 1, whereby 2'-O-(fi-D- glucopyranosyl)-D-pantothenic acid wasobtained. The N.M.R. on the product gave the same results as in Example1.

EXAMPLE 4 Ethyl 4' O acetyl 2' O (O-acetyl-fl-cellobiosyD-D-pantothenate was prepared and isolated in the same manner as inExample 1 except that 4.9 g. of ethyl 4'-O- acetyl-D-pantothenate, 4.3g. of Hg. (CN) and 12 g. of hepta-O-acetyl-a-cellobiosyl bromide wereused. Yield was 38%.

The N.M.R. in CD01 on the resultant product gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.89 (3H, s), 0.97 (3H, s), 1.26 (3H, t, I=7 c.p.s.), 2.0-

2.2 (7 x CH 2.55 (2H, t, J=7 c.p.s.), 3.5-5.3 (m), 6.8 (1H, t).

0.88 (6H, s), 2.59 (2H, t, J=7 c.p.s.), 3.20-4.00 (m),

EXAMPLE 5 6.0 g. of benzyl 2'-O-benzyl-D-pantothenate was dissolved in50 ml. of a 2:1 volume ratio dried mixture of 10 nitromethane andbenzene. To the solution were added 5.1 g. of dried Hg(CN) and 10 g. ofactivated CaSO in the form of powder and the mixture was stirred at roomtemperature for 1 hour. To the mixture was further added 8.2 g. oftetra-O-acetyl--D-glucopyranosyl bromide and the mixture was refluxedfor 10 hours.

After being cooled, the resultant reaction mixture was filtered and theresidue was washed with benzene. The washed liquid was mixed with thefiltrate and the mixture was concentrated under reduced pressure. Theconcentrated liquid was dissolved in benzene to produce 100 m1. ofbenzene solution and the solution was washed with aqueous potassiumbromide solution and then with saturated sodium hydrogen carbonatesolution and finally with water. The washed liquid was dried with K COand concentrated under reduced pressure, whereby oily substance,yellowish brown in color, was obtained. The oily substance was thensubjected to silica gel column chromatography to produce 6.0 g. ofbenzyl 2-O-benzyl-4-O-(O- acetyl B D glucopyranosyl) D pantothenate.Yield was 40%.

The N.M.R. in CDCl;, on the resultant product gave the followingresults.

N.M.R. spectrum (6, p.p.m.):

0.87 (3H, s), 0.94 (3H, s), 1.98 (4 x CH 2.53 (2H, t, J=6 c.p.s.),3.1-4.7 (m), 5.05 (2H, s), 7.25 (10 H).

6.0 g. of benzyl2'-0-benzyl-4'-O-(O-acetyl-B-D-glucopyranosyl)-D-pantothenate wasdissolved in 30 ml. of acetic acid and then 1.0 g. of palladium blackwas added to the solution. The resultant mixture was violently stirredin a hydrogen gas atmosphere at room temperature for 8 hours forcatalytic reduction. After being filtered and concentrated the resultantreaction mixture was subjected to silica gel column chromatography usinga 95 :4:1 volume ratio mixture of CHC13, CH OH and CH COOH or a 95:5volume ratio mixture of CHCl and C H OH, and thereafter recrystallizedfrom ether, whereby 2.3 g. of 4'- O-(tetra-O-acetyl-fi-D-glucopyranosyl)-D-pantothenic acid was obtained. Yield was The N.M.R. in CDCl on theresultant product gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.91 (3H, s), 0.97 (3H, s), 2.0-2.1 (4x CH 2.6 (2H, t,

J=6 c.p.s.), 3.1-5.3 (m), 7.00 (1H, m).

2.3 g. of 4'-O-(tetra-O-acetyl-fl-D-glucopyranosyl)-D- pantothenic acidwas dissolved in 30 ml. of dried metha nol. To the solution was added 15ml. of 0.4 N Ba(OCH solution dissolved in methanol while being cooledwith ice water and the resultant mixture was left to stand at 0 C. for24 hours. To the resultant reaction mixture was added 20 ml. of waterand the mixture was passed through a column of Dowex l x 8 (trademark,the same as in Example 1). After being washed with water the product waseluted with 0.5 N acetic acid and freeze-dried to obtain 1.5 g. of4'-O-(B-D-glucopyranosyl)- D-pantothenic acid as a white hygroscopicpowder. Yield was 94%.

The N.M.R. in D 0 on the resultant product and elementary analysisthereof gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.88 (3H, s), 0.94 (3H, s), 2.58 (2H, t, J=6 c.p.s.), 3.2-3.9 (m), 4.03(1H, s), 4.37 (1H, d, J=6 c.p.s.).

Elementary analysis (percent): Found: C, 47.29; H, 7.12; N, 3.80. Calcd.for C15H27O10NZ C, 47.24; H, 7.14; N, 3.67.

EXAMPLE 6 Benzyl 2' O benzyl 4-O-(O-acetyl-p-cellobiosyl)-D pantothenatewas prepared and isolated in the same man nor as in Example 5 exceptthat 4.0 g. of benzyl 2'-O- benzyl-D-pantothenate,. 2.6 g. of Hg(CN)powder and 1 1 7.0 g. of hepta-O-acetyl-a-cellobiosyl bromide were used.Yield was 40%.

The N.M.R. in CD01 on the resultant product gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.85 (3H, s), 0.94 (3H, s), 2.02 (7 x CH 2.53 (2H,

t, J-=6 c.p.s.), 3.1-5.2 (In), 7.27 (10 H).

1.5 g. of benzyl2-O-benzyl-4'-O-(O-acetyl-fl-cellobiosyl)-D-pantothenate was dissolvedin 10 ml. of ethanol and to the solution was added 0.3 g. of palladiumblack. The mixture was subjected to catalytic reduction in the samemanner as in Example 5 to produce 0.7 g. of 4'-0- (O acetyl Bcellobiosyl)-D-pantothenic acid havin a melting point of 100 to 115 C.Yield was 85%.

The N.M.R. in CDCl on the resultant product gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.91 (3H, s), 0.95- (3H, s), 2.03 (7 x CH 2.57 (2H, m),

0.55 g. of 4-O-(O-acetyl-p-cellobiosyl)-D-pantothenic acid was dissolvedin 20 ml. of dried methanol. To the solution was added 2.5 ml. of 0.4 NBa(CH solution dissolved in methanol while being cooled with ice waterand the resultant mixture was left to stand at 0 C. for 24 hours. Theresultant reaction mixture was treated in the same manner as in Example1, whereby 330 mg. of 4'-'O-( 3-cellobiosyl)-D-pantothenic acid wasobtained. Yield was 93%.

The N.M.R. in D 0 on the resultant product and elementary analysisthereof gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.89 (3H, s), 0.92 (3H, s), 2.58 (2H, t, J=6 c.p.s.), 3.2-3.9, m), 4.02(1H, s), 4.36 (1H, d, J=6 c.p.s.), 4.48 (1H, d, 1:6 c.p.s.).

Elementary analysis (percent): Found: C, 46.60; H,

7.00; N, 2.57. Calcd. for C H O N-C H OH (percent) C, 46.85; H, 7.35; N,2.38.

EXAMPLE 7 4.0 g. of benzyl 2'-O-benzyl-D-pantothenate, 2.8 g. of Hg(CN)and 10 g. of CaSO, were added to 40 ml. of a 3 :1 volume ratio mixtureof nitromethane and benzene, and the mixture was stirred at roomtemperature for 2 hours. To the resultant solution was added 4.8 g. oftri- O-benzoyl-D-ribofuranosyl chloride dissolved in 20 ml. of benzeneand the resultant mixture was then slowly refluxed for 20 hours withstirring.

After being cooled, the resultant reaction mixture was filtered and thefiltrate was concentrated under reduced pressure. The concentratedliquid was then dissolved in benzene and the benzene solution obtainedwas washed with 1 M of KBr and then with water and concentrated underreduced pressure. The concentrated product was subjected to silica gelcolumn chromatography with 1% ethanol-containing CHC1 to obtain 8.7 g.of benzyl 2'- O-benzyl-4-O-(tri-O-benzoyl-fi D ribofuranosyD-D-pantothenate, a somewhat impure condensation product.

The product was then subjected to catalytic reduction in ml. of aceticacid in the presence of H gas using 1 g. of palladium black as acatalyst. The resultant product was thereafter subjected to silica gelcolumn chromatography with a 96:3:1 volume ratio mixture of CHClmethanol and acetic acid. The resulting substance was subsequentlyrecrystallized from ether-petroleum ether to obtain 5.2 g. of4'-O(tri-O-benzoyl-p-D-ribofuranosyl)-D-pantothenic acid. Yield was 78%.

The N.M.R. in CDCl on the resultant product and elementary analysisthereof gave the following results.

N.M.R. spectrum (6, p.p.m.):

Elementary analysis (percent): Found: C, 63.32; H, 5.69; N, 2.17. Calcd.for C35H37012N: C,63.34; H, 5.62; N, 2.11.

3.2 g. of the product thus obtained was dissolved in 10 ml. of a 1:4volume ratio mixture of CH CI and methanol and the solution was cooledto 0 C., whereupon 15 ml. of 0.5 N NaOCH Was added to the solution. Themixture was then left standing at room temperature for 30 hours. Afterneutralization and subsequent concentration under reduced pressure, theresultant product was made into 30 ml. of an aqueous solution, fromwhich benzoic acid was removed by extraction with ether. The product wasthen applied to a Dowex 1 x 8 (trademark, the same as in Example 1)column and then washed with water, this being followed by elution with0.5 N acetic acid and freeze-drying to give 1.6 g. of oily4-O-(fl-D-ribofuranosyl) D pantothenic acid. Yield Was 94%.

The N.M.R. in D 0 on the product and elementary analysis thereof gavethe following results.

N.M.R. spectrum (6, p.p.m.):

0.94, 0.99, 1.02, 2.58 (2H, t, J=7 c.p.s.), 3.25-3.95 (m),

4.02 (s), 4.05-4.32 (In), 4.97.

Elementary analysis (percent): Found: C, 47.29; H, 7.28; N, 3.88. Calcd.for C H O N: C, 47.86; H, 7.17; N, 3.99.

EXAMPLE 8 In the same manner as in Example 5, benzyl 2'-O- benzyl-4-O-(Oacetyl B D galactopyranosyl)-D- pantothenate was prepared, which wassubjected to silica gel column chromatography with a 6:4 volume ratiomixture of benzene and CH COOC H to obtain 2.15 g. of a purified product'by using 2.0 g. of benzyl 2-O-benzyl- D-pantothenate, 1.9 g. of Hg(CN)and 3.5 g. of tetra- O-acetyl-a-D-galactopyranosyl bromide. Yield was59%.

2.15 g. of benzyl 2' O benzyl-4'-O-(O-acetyl-fl-D-galactopyranosyl)-pantothenate thus prepared was dissolved in 5 ml. ofacetic acid, and 0.2 g. of palladium black was added to the resultingsolution. The solution was then subjected to catalytic reduction in thesame manner as in Example 5 and subsequently purified by silica gelchromatography with 5% ethanol-containing CHCl to obtain 1.5 g. of4-O-(O-acetyl-fi-Dgalactopyranosyl)-D-pantothenic acid. Yield was 93%1.5 g. of this product was dissolved in 30 ml. of dry methanol, and 10ml. of 0.4 N Ba(OCH was added to the solution while the solution waskept at 0 C. The mixture was left to stand at 0 C. for 24 hours. Thereaction solution was treated in the same manner as in Example 5 toobtain 850 mg. of 4'-O-(B-D-galactopyranosyl)-D-pantothenic acid. Yieldwas 82%.

The N.M.R. in D 0 on the resulting product and elementary analysisthereof gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.87 (3H, s), 0.93 (3H, s), 2.58 (2H, t, J =7 c.p.s.), 3.3-

4.0 (m), 4.04 (1H, s), 4.32 (1H, (1, 1:7 c.p.s.).

Elementary analysis (percent): Found: C, 47.40; H, 7.17; N, 3.64. Calcd.for C H O N: C, 47.24; H, 7.14; N, 3.67.

EXAMPLE 9 In the same manner as in Example 5, benzyl 2'-O-benzyl-4-O-(O-acetyl 18 maltosyl)-D-pantothenate was prepared, which wasthen purified by silica gel column chromatography with 2.5%ethanol-containing CHCl to obtain 3.1 g. of a purified product, using3.2 g. of benzyl 2'-O-benzyl-D-pantothenate, 2.1 g. of Hg(CN) powder and5.6 g. ofhepta-O-acetyl-a-maltosyl bromide. Yield was 38%.

3.1 g. of the benzyl2'-O-benzyl-4-O-(O-acetyl-fimaltosyl)-D-pantothenate was dissolved in 7ml. of acetic acid, and 0.2 g. of palladium black was added to thesolution. In the same manner as in Example 5, the resulting solution wassubjected to catalytic reduction and subsequently to silica gel columnchromatography with a 95 :4:1 volume ratio mixture of CHCl CH OH and CHCOOH to obtain 1.6 g. of 4'-O-(O-acetyl-fi maltosyl)-D-pantothenic acid.Yield was 63%.

1.2 g. of this product was dissolved in 50 ml. of dry methanol, and thesolution was cooled to C., whereupon 4 ml. of 0.5 N NaOCH was added tothe solution. The mixture was left to stand at room temperature for 3hours. After addition of 20 ml. of H 0, the reaction product waspurified by a Dowex 1 x 8 (trademark, the same as in Example 1) columnin the same manner as in Example 5. The purified product was thensubjected to freeze-drying to give 700 mg. of 4O-(,B-maltosyl)-D-pantothenic acid. Yield was 89%.

The N.M.R. in D 0 on the resulting product and elementary analysisthereof gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.89 (3H, s), 0.94 (3H, s), 2.58 (2H, h, I=7 c.p.s.), 3.2-4.0 (m), 4.01(1H, s), 4.37 (1H, d, 1:7 c.p.s.), 5.31 (1H, (1, 1:3 c.p.s.).

Elementary analysis (percent): Found: C, 46.48; H, 6.88; N, 2.54. Calcd.for C H O' N: C, 46.40; H, 6.86; N, 2.58.

EXAMPLE 10 To 30 ml. of dried benzene were added 3.35 g. of methylD-pantothenate and 4.0 g. of Hg(CN) To the resultant mixture was addeddropwise in 6 hours with stirring under reflux condition 10 g. of2,3,4,6-tetra-O- benzoyl-a-D-glucopyranosyl bromide dissolved in 100 ml.of benzene. The reaction system was further refluxed with stirring for 2hours.

After being cooled the resultant reaction mixture was filtered. Theresidue was washed with benzene and after the washing was mixed with thefiltrate the mixture was subjected to concentration under reducedpressure. The concentrate was dissolved in 100 ml. of benzene and washedwith aqueous potassium bromide solution, then with saturated sodiumhydrogen carbonate solution and finally with water. The washed benzenesolution was dried with potassium carbonate and concentrated underreduced pressure. The concentrate was subjected to silica gel columnchromatography using chloroform containing 2.5 vol. percent of ethanol,whereby 4.0 g. of methyl 4'-O- (2,3,4,6-tetra-O-benzoyl-8-D-glucopyranosyl) D pantothenate was obtained.

The N.M.R. in CDCl on the resultant product and elementary analysisthereof gave the following results.

N.M.R. spectrum (6, p.p.m.):

0.85 (6H, s), 2.44 (2H, t, I=7 c.p.s.), 3.23-3.95 (m), 4.50-4.95 (m),6.02-5.57 (m), 7.20-8.10 H, m).

Elementary analysis (percent): Found: C, 64.96; H, 5.65; N, 1.77. Calcd.for C H O N: C, 65.09; H, 5.59; N, 1.73.

1.6 g. of methyl 4'-O-(2,3,4,6-tetra-O-benzoyl-fl-D-glucopyranosyl)-D-pantothenate was dissolved in 5 ml. ofdichloromethane, to which 15 ml. of dried methanol was further added. 6ml. of 0.5 N NaOCH was added to the solution and thereafter the solutionwas left to stand at 20 C. for 24 hours. After being cooled andneutralized with hydrochloric acid the resultant reaction mixture waspassed through a column of Dowex 1 x 8 (trademark, the same as inExample 1). After being washed with water the product was eluted with0.5 N acetic acid and freezedried to produce 0.68 g. of4-O-(B-D-glucopyranosyD- D-pantothenic acid in the form of whitehygroscopic powder. Yield was 90%.

The N.M.R. spectrum on the resultant product in D 0 and elementaryanalysis thereof gave the following results.

14 N.M.R. spectrum (6, p.p.m.):

0.88 (3H, s), 0.94 (3H, s), 2.58 (2H, t, I=6 c.p.s.), 3.2-3.9 (m), 4.03(1H, s), 4.37 (1H, d, J=6 c.p.s.). Elementary analysis (percent): Found:C, 47.28; H,

7.11; N, 3.80. Calcd. for C H O N: C, 47.24; H, 7.14;

EXAMPLE 11 To 50 ml. of dried benzene were added 7.0 g. of methylD-pantothenate and 8.5 g. of Hg(CN) To the resultant mixture was addeddropwise in 10 hours with stirring under reflux condition 13.0 g. of2,3,4,6-tetra-O-acetyl-a- D-glucopyranosyl bromide dissolved in ml. ofbenzene, and the reaction system was further refluxed with stirring for2 hours.

The resultant reaction mixture was treated in the same manner as inExample 10 and subjected to silica gel column chromatography usingchloroform containing 4.0 vol. percent of ethanol, whereby 3.27' g. ofmethyl 4'-O- (2,3,4,6-tetra-O-acetyl-fl-D-glucopyranosyl) D pantothenatewas obtained.

N.M.R. in CDCl on the resultant product and elementary analysis thereofgave the following results.

N.M.R. spectrum (6, p.p.m.):

0.90 (3H, s), 0.98 (3H, s), 1.96-2.12 (4X CH CO), 2.55

(2H, t, 1:6 c.p.s.), 3.20-5.20 (m), 3.68 (3H, s). Elementary analysis(percent): Found C, 51.11; H,

6.67; N, 2.49. Calcd. for C H O N: C, 51.15; H, 6.62;

200 mg. of methyl 4'-O-(2,3,4,6-tetra O acetyl-fi-D-glucopyranosyl)-D-pantothenate was dissolved in 5 ml. of methanol. 1 ml.of 0.4 N Ba(OCH was added to the solution cooled with ice and thereafterthe solution was left to stand at 0 C. for 24 hours.

After diluted with 10 ml. of water the resultant reaction mixture wastreated in the same manner as in Example 10, whereby 127 mg. of4'-O-(fl-D-glucopyranosyD- D-pentothenic acid was obtained.

N.M.R. in CDCl on the resultant product and elementary analysis thereofgave the following results.

N.M.R. spectrum (6, p.p.m.):

0.88 (3H, s), 0.94 3H, s), 2.58 (2H, t, J=6 c.p.s.), 3.2-3.9 (m), 4.03(1H, s), 4.37 (1H, d, J 6 c.p.s.). Elementary analysis (percent): Found:C, 47.27; H,

7.12; N, 3.78. Calcd. for C H O N: C, 47.24; H, 7.14;

What we claim is:

1. A glycosyl pantothenic acid having a formula of wherein one of R andR is hydrogen and the other is fi-D-ribofuranosyl, 3 D glucopyranosyl,B-D-galactopyranosyl, ,B-cellobiosyl or B-maltosyl.

2. The glycosyl pantothenic acid according to claim 1, in which R ishydrogen and R is fl-D-glucopyranosyl or fl-cellobiosyl.

3. The glycosyl pantothenic acid according to claim 1, in which R is8-Dribofuranosyl, fi-D-glucopyranosyl, B- D-galactopyranosyl,fi-cellobiosyl or ,B-maltosyl, and R is hydrogen.

4. The glycosyl pantothenic acid according to claim 3, in which said Ris fi-D-glucopyranosyl.

References Cited UNITED STATES PATENTS 3,356,674 12/1967 Ikeda et al.260-210 R 3,380,992 4/ 1968 Argoudelis et al. 260-210 R JOHNNIE R.BROWN, Primary Examiner U.S. Cl. X.R.

